PROJECT SUMMARY Over the past two decades, the development of new immunosuppressive agents has greatly improved early post-transplant survival. Nevertheless, long-term survival has not improved significantly, due to chronic rejection, infections, and malignancies - all attributable to chronic immunosuppression. In lung transplantation, chronic allograft rejection, as manifested by obliterative bronchiolitis, fibrosis, and/or vasculopathy, represents the principal cause of graft loss and patient demise after the first year following transplantation. It is now well established that both antigen-dependent and antigen-independent factors contribute to both acute and chronic rejection in a synergistic manner. We have long held the position that the establishment of transplant tolerance is the paradigm shift that will transform the field of transplantation by obviating the need for long term systemic immunosuppression with its attendant complications (malignancy, toxicity, and infection), while simultaneously preventing both acute and potentially chronic rejection. Tolerance of kidney allografts has been achieved in non-human primates (NHPs) and in humans using a strategy that induces hematopoietic mixed chimerism. We have modified and extended this approach to lung transplantation in NHPs by adding the interleukin (IL)-6 signal blocker tocilizumab (anti-IL6R mAb) to our delayed, non-myeloablative conditioning regimen. This has allowed us to achieve long-term lung allograft tolerance without chronic rejection in NHPs for the first time, albeit only in one-MHC haplotype mismatched donor/recipient pairs. Attempts to cross more stringent histocompatibility barriers have led to acute and/or chronic rejection. From a mechanistic point of view, it appears that the tolerance obtained in NHPs is not simply deletional tolerance, as seen in most murine chimeric models, but is highly dependent on 1) enhancing regulatory responses and 2) dampening pro- inflammatory states. We therefore hypothesize that to successfully induce tolerance of fully MHC-mismatched lung allografts without the development of chronic rejection we must develop protocols which further enhance immune regulation and which prevent antigen-independent inflammation. This hypothesis will be tested in Aim 1. Our overall goal is to generate a tolerance protocol that can be applied to human lung allograft recipients. Thus, once the optimal tolerance induction protocol is identified in Aim 1, attempts will be made to substitute the investigational components of the protocol with FDA-approved agents making it suitable for clinical use in Aim 2. Finally, it is clear that chronic rejection is refractory to current therapies, including in some circumstances, tolerance induction. Therefore, in Aim 3 we will perform integrated mechanistic studies of the pathogenesis of chronic rejection and use the results to design modifications of the optimal protocol achieved in Aim 2 in order to prevent this complication.